This disclosure relates to methods and kits for detecting a subject""s sensitivity to pharmaceutical agents, particularly an animal""s sensitivity to application of drugs (such as ivermectin) that interact with P-glycoprotein. It also relates to variants of the mdr1 gene, which variants impact transport of drugs that interact with the P-glycoprotein, as well as cell and whole animal systems comprising such variants and methods of using these systems.
The observation, over 100 years ago, that certain chemical dyes injected into the peripheral circulation were able to gain access to most organs but not the brain led to the concept of a blood-brain barrier. Research in the 1960""s demonstrated that the anatomical basis of the blood-brain barrier is the specialized endothelial cells of brain capillaries. While it has been thought that the entry of drugs, toxins, and xenobiotics into the brain is simply a function of lipophilicity, electrical charge, and molecular weight, ongoing research demonstrates that the capillary endothelium composing the blood-brain barrier is not simply an anatomic entity. A number of active transport systems exist that selectively regulate both influx and efflux of compounds across brain capillary endothelial cells. The most important drug-efflux system of the blood-brain barrier identified to date is P-glycoprotein.
P-glycoprotein, the product of the mdr1 (multidrug resistance) gene, is a 170-kD membrane-spanning, cell-surface protein that functions as a drug-efflux pump. P-glycoprotein was first identified over 20 years ago in chemotherapeutic drug-resistant tumor cells, and is now known to be a major cause of multidrug resistance in human and veterinary cancer patients. In tumor cells, P-glycoprotein functions as an ATP-dependent efflux pump resulting in decreased intracellular drug accumulation and reduced cytotoxicity. Chemotherapeutic drugs that are substrates for P-glycoprotein include Vinca alkaloids (vincristine and vinblastine), doxorubicin and related compounds, taxanes, and epipodophyllotoxins. Alkylating agents, platinum compounds, and antimetabolites are not substrates for P-glycoprotein. Though these agents are structurally and functionally dissimilar, P-glycoprotein substrates share several other characteristics. They typically are complex, hydrophobic, amphipathic compounds that are natural products (i.e., derived from plants or micro-organisms) or analogs of natural products. A number of non-cytotoxic compounds have been identified as P-glycoprotein substrates, including steroid hormones, bilirubin, antiparasitic agents, selected antimicrobial agents, and others.
P-glycoprotein is expressed not only in tumor cells, but also in a variety of normal tissues, including renal tubular epithelium, canalicular surfaces of hepatocytes, adrenal cortical cells, colonic and intestinal epithelium, placenta, apical margins of bronchiolar epithelium, and brain capillary endothelial cells. Consistent with its function as a transport pump, the expression of P-glycoprotein in non-neoplastic tissues suggests a normal physiologic role for P-glycoprotein mediating the export of potentially toxic xenobiotics from the body. Although the normal function of P-glycoprotein in many of these tissues has not been elucidated, a great deal is known about its role in the blood-brain barrier.
Avermectins are a class of natural products with broad antiparasitic activity. Ivermectin, a semi-synthetic lactone in the avermectin family, is a drug that is used extensively in veterinary medicine to treat and control infections caused by nematode and arthropod parasites. It is also used in human medicine to treat onchocerciasis, lymphatic filariasis, and strongyloidiasis. Ivermectin induces a tonic paralysis in invertebrate organisms by potentiating glutamate-gated chloride channels, and/or gamma-amino butyric acid (GABA)-gated chloride channels (Tracy and Webster, In: Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9th edition. Hardman et al., eds. New York: McGraw-Hill, 1996: 1009-1026, 1996) of the peripheral nervous system. In most mammals, the blood-brain barrier prevents access of ivermectin to the central nervous system, and since GABA receptors in mammals are restricted to sites within the central nervous system, mammals are generally protected from the neurologic effects of ivermectin (Fisher and Mrozik, Annu. Rev. Pharmacol. Toxicol. 32:537-553, 1992).
There are some specific subgroups of mice and dogs, however, that are exquisitely sensitive to the neurologic actions of ivermectin. Genetically engineered mdr1a knock-out [mdr1a(-/-)] mice are 50 to 100 times more sensitive to ivermectin-mediated neurotoxicity than wild-type mice (Schinkel et al., Cell. 77:491-502, 1994), and accumulate 80-90-fold higher concentrations of ivermectin in the brain than do wild-type mice. The protein product of mdr1a, called P-glycoprotein (P-gp) is a 170-kD transmembrane protein pump that is present at high concentrations in the apical membrane of brain capillary endothelial cells (Van Asperen et al., J. Pharmaceut. Sci. 86:881-884, 1997, 1997; Tsuji, Therap. Drug Monitor. 20:588-590, 1998). Substrates of P-gp include a variety of large, structurally unrelated hydrophobic compounds, including naturally occurring compounds such as ivermectin, cyclosporin, digoxin, and others. After substrates are bound by P-gp, they are actively extruded from the endothelial cell into the capillary lumen (Van Asperen et al., J. Pharmaceut. Sci. 86:881-884, 1997). Abrogation of P-gp results in failure of the blood-brain barrier. High concentrations of ivermectin accumulate in brain tissue from mdr1a (-/-) mice, and neurotoxicity ensues.
Approximately 25% of a population of the CF-1 mouse strain were much more sensitive to neurotoxicity produced by ivermectin than unaffected mice of the same strain (Umbenhauer et al., Toxicol Appl. Pharmacol. 146:88-94, 1997). Investigation into the cause of this sensitivity led to the discovery that the sensitive animals did not express P-gp in their brain endothelial cells. Furthermore, a restriction-fragment-length polymorphism in the murine mdr gene was documented that allowed prediction of sensitive animals, and an inheritance pattern following normal Mendelian genetics was observed (Umbenhauer et al., Toxicol Appl. Pharmacol. 146:88-94, 1997).
In dogs, a breed-related sensitivity to ivermectin has been reported in Collies, that may affect 30 to 50% of the population (Pulliam et al., Veter. Med. 7:33-40, 1985; Hsu et al., Comp. Contin. Educat. Veter. Pract. 11:584-589, 1989, Paul et al., Am. J. Vet. Res. 48:685-688, 1987). In one study, 1/200th of the lethal dose of ivermectin for Beagles was lethal for Collies (Pulliam et al., Veter. Med. 7:33-40, 1985). Other related canine breeds believed to be affected by ivermectin sensitivity include Border Collies, Shetland Sheepdogs, Old English Sheepdogs, and Australian Shepherds (Campbell and Benz, J. Vet. Pharmacol. Therap. 7:1-16, 1984).
Despite numerous investigations (Vaughn, et al., Vet. Res. Commun. 13:47-55, 1989; Roher et al., Vet. Res. Commun. 14:157-165, 1990; Pulliam et al., Veter. Med. 7:33-40, 1985), the mechanism for ivermectin-sensitivity in Collies is unknown.
The disclosure provides a mutation in the mdr1 gene, which results in production of truncated and non-functional P-gp and thereby causes sensitivity to ivermectin and other drugs that serve as P-gp substrates. With the identification of this mutation, methods are provided to determine if an individual subject is sensitive to ivermectin. Also provided are systems for examining the importance of P-gp in drug transport in whole animal and cell culture systems.
A provided embodiment is a method of detecting ivermectin sensitivity in a subject (for instance a mammal, such as a canine animal), which method includes determining whether a gene-truncation mutation in a mdr1-encoding sequence of the subject or a truncated P-pg is present in the subject. Such a gene-truncation mutation or truncation of P-gp indicates that the subject is sensitive to ivermectin. In specific examples of such methods, the gene truncation mutation is a deletion of about four base pairs at about residue 294-297 of SEQ ID NO: 1 (the canine mdr1 cDNA) or a homologous cDNA or gene.
In certain embodiments, methods provided herein are used to evaluate whether the subject can be treated safely with ivermectin or another drug that can be excluded from the brain by P-gp (such as those listed in Table 2).
In certain provided methods, the method includes determining whether the subject is homozygous or heterozygous for the gene-truncation mutation.
In specific examples of the provided methods, determining whether a gene-truncation mutation is present in the subject includes subjecting DNA or RNA from the subject to amplification using oligonucleotide primers, for instance in performing an oligonucleotide ligation assay.
In a specific embodiment provided herein, the method of detecting ivermectin sensitivity in a subject involves obtaining a test sample of DNA containing a mdr1 sequence of the subject; and determining whether the subject has the gene-truncation mutation in the mdr1 sequence, wherein the presence of the mutation indicates sensitivity to ivermectin. In certain examples of this embodiment, determining whether the subject has the mutation comprises using restriction digestion, probe hybridization, nucleic acid amplification, or nucleotide sequencing.
Further embodiments of methods provided herein involve obtaining from the subject a test sample of DNA comprising an mdr1 sequence; contacting the test sample with at least one nucleic acid probe for an mdr1 gene truncation mutation that is associated with ivermectin sensitivity, to form a hybridization sample; maintaining the hybridization sample under conditions sufficient for specific hybridization of the mdr1 sequence with the nucleic acid probe; and detecting whether the mdr1 sequence specifically hybridizes with the nucleic acid probe, wherein specific hybridization of the mdr1 sequence with the nucleic acid probe indicates ivermectin sensitivity. In specific examples of such embodiments, the probe is present on a substrate, for instance a nucleotide array.
Also provided are methods of detecting ivermectin sensitivity in a subject by determining whether truncated P-gp is present in a sample from the subject. Certain examples of such methods will involve reacting at least one P-gp molecule contained in the sample from the subject with a P-gp-specific binding agent (such as an antibody) to form a P-gp:agent complex. Such methods can further include detecting the P-gp:agent complex, for instance by Western blot assay, ELISA, or other immunoassay technique.
Also provided herein are kits for use in diagnosing ivermectin sensitivity in a subject. Such kits include at least one probe that specifically hybridizes to an mdr1 gene-truncation mutation associated with ivermectin sensitivity. In specific examples of such kits, the probe specifically hybridizes to an mdr1 gene-truncation mutation at or about residue 294-297 of SEQ ID NO: 1.
Other provided kits for use in diagnosing ivermectin sensitivity in a subject contain a P-gp-specific binding agent, such as an antibody. In specific examples of such kits, the provided agent is capable of specifically binding to truncated P-gp protein.
Also provided herein are oligonucleotides that specifically hybridize to a canine mdr1 gene-truncation mutation, for instance an oligonucleotide that hybridizes to an mdr1 gene-truncation mutation at residue 294-297.
Other embodiments are systems and methods for studying the effects of drugs (and drug candidates) on biological systems expressing a mdr1 gene truncation, such as the mdr1 gene-truncation mutation at residues 294-297. Examples of such systems include cultured cells (such as intestinal epithelial cells, brain endothelial cells (for instance, capillary endothelial cells), renal-tubular cells, hepatocytes, or neoplastic cells) isolated from a canine that naturally exhibits a gene truncation mutation in the mdr1 gene. Other examples of such systems include animal models (including for instance dogs) in which the mdr1 gene is naturally truncated, or in which such truncations have been engineered using recombinant technologies and/or cloning. Methods are also provided for using these animal models and cell systems, for instance to study drug interactions with P-gp or to examine the impact of drugs and drug candidates on biological systems. Such methods would be particularly useful in the drug approval process.
One embodiment is a method of determining a P-gp influenced biological effect of a compound on a canine cellular system, which method involves contacting a canine cell having a truncation mutation in its mdr1 gene with the compound, and comparing a characteristic (such as a genetic, physiological, chemical, or morphological characteristic) of the canine cell contacted with the compound with the characteristic of a similar canine cell that was not contacted with the compound. In such methods, a difference in the characteristic between the two cells is indicative of the P-gp influenced biological effect in the cell. In specific examples of such methods, the canine cell is a Collie cell. The truncation mutation in the mdr1 gene is in some examples a mutation at residue 294-297.
Specific types of canine cells include, but are not limited to, gastrointestinal tissue cells, renal tissue cells, nerve tissue cells, brain capillary endothelial cells, and liver tissue cells. In some methods, the canine cell is a neoplastic cell.
Also provided are methods of determining a P-gp influenced biological effect of a compound on a canine cellular system, wherein contacting the canine cell with the compound occurs in vivo in the native environment of the canine cell, for instance in a dog (such as a Collie dog).
In some examples of the provided methods, biological effects include absorption or distribution of a drug or compound, for instance a drug or compound that interacts with or is transported by P-gp.
Also provided is an animal model useful for studying a P-gp influenced biological effect of a compound, comprising a Collie identified as being homozygous or heterozygous for a truncation mutation in the mdr1 gene (for instance, a mutation at residue 294-297). Also provided are methods of using this animal model to examine the effect of compounds that interact with P-gp, for instance compounds that are transported by P-gp or that modulate its transport activity.
Compounds contemplated for use in the methods provided herein include anti-infective agents (e.g., antiviral, antibacterial, or anti-prion agents), antineoplastic agents, analgesics, neurokinin receptor antagonists, anti-emetic agents, beta-adrenergic receptor antagonists, antiepileptic agents, anti-psychotic agents, anti-depressive agents, and other drugs that act on the central nervous system.